[Histonet] RE: fixing cultured cells on coverslips

Pixley, Sarah (pixleysk) PIXLEYSK <@t> UCMAIL.UC.EDU
Wed Jul 20 15:48:21 CDT 2005

Dear LaCinda:

For lightly attached cells grown on glass coverslips, like neurons, we have
found the fixation protocol below will help (but not always!) keep them on
the slip through IHC. Two things were important: 1) having the fixative at
the same temperature (and pH) as the cells so that they didn't shrink from
the temp (or pH) change and 2) not rinsing until after they are fixed.
Delicately attached cells do not do well when exposed to air during a medium

1.Warm 4% paraformaldehyde (pH 7.4 in 0.1 M phosphate buffer) to 37 degrees
2. Add small volume of 4% PF directly into the medium that the cells are
growing in. (In other words, don't remove the culture medium so that you
don't expose the cells to an air-water interface.) Use approximately the
same volume as the cells are growing in, so that you have roughly 2% para.
Incubate 5 minutes.
3. Remove all the solution over the cells after the 5 minutes. Add fresh 4%
para. Incubate 10 minutes.
4. Gently wash 3 times with room temperature PBS or PB. Keep being gentle
during all rinses. Large bore plastic transfer pipets are what we currently

I would also like to know about a list serve for culture people.
Best of luck!
Sarah Pixley
Univ. Cincinnati College of Med.

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